Cardiac safety pharmacology is the study of the potential undesirable pharmacodynamic effects of a substance on heart function in relation to exposure to the substance in the therapeutic range and above. Cardiac safety is a major concern in current drug development. Since 1981, at least 10 blockbuster drugs have been withdrawn from the market due to cardiac liability, defined as potentially undesirable effects on heart function. Furthermore, cardiac safety is a major reason for late stage attrition of drug candidates during development.
There are three non-mutually exclusive ways that non-cardiac drugs may lead to cardiac liability. Directly, cardiotoxic drugs are drugs which cause damage via necrosis or apoptosis, such as anthracyclines. Pro-arrhythmic drugs are drugs which induce arrhythmia. Indirectly, cardiotoxic drugs are drugs which indirectly affect cardiac function, such as by causing narrowing of the arteries.
Directly, cardiotoxic drugs directly affect the viability of cardiomyocytes and therefore heart function. A prominent class of drugs in this category is chemotherapeutic drugs, such as anthracyclines. Mortality due to cardiac disease is thought to be 8-fold higher for survivors of childhood cancers who have received chemotherapy. These drugs are thought to disrupt iron metabolism, generating harmful oxygen radical species which ultimately cause mitochondrial damage and apoptosis.
Pro-arrhythmic drugs induce arrhythmia. Normal synchronized contractile activity of cardiomyocytes is the result of orchestrated ion currents passing across the cell membrane via ion-specific channels and coupling with the specialized cytoskeleton. Disturbances in the ionic movement of interference with ion channel activities may lead to arrhythmia. It is believed that one of the primary targets of pro-arrhythmic drugs is the ERG channel, which is responsible for delayed repolarization of cardiomyocytes. ERG channel blockage may lead to QT elongation and this may cause a fatal form of ventricular arrhythmia called Torsades de Pointes (TdP). Between 1990 and 2006, 10 blockbuster drugs have been withdrawn from the market due to induction of TdP. The drugs that have been associated with cardiac arrhythmia and removed from the market are prenylamine, terodiline, sparfloxacin, sertindole, terfenadine, astemizole, grepafloxacin, cisapride, droperidol, and levacetylmethadol.
Excitation-contraction coupling (ECC) is a term used to describe the physiological process of converting an electrical stimulus to a mechanical response. The process is fundamental to muscle physiology, wherein the electrical stimulus may be an action potential and the mechanical response is in the form of contraction. Although ECC has been known generally over half a century, it is still an active area of biomedical research.
Cardiomyocytes are specialized muscle cells of the myocardium that are capable of excitation-contraction coupling. Cardiomyocytes are commonly used in biomedical research to assess the cardiotoxicity of potential drugs or treatments. Two conventional approaches to assess cardiotoxicity are primarily used. A first approach involves isolation of cardiomyocytes directly from a mammalian species such as rats and dogs followed by electrophysiological studies on the isolated cardiomyocytes. However, this approach suffers from being extremely labor-intensive, time consuming and costly and at the same time not very amenable to the high throughput demands of pharmaceutical industry. An alternative approach utilizes cell-based assay models, which heterologously express specific ion channels such as hERG channels or voltage-gated calcium channels. These cardiac ion channels have been envisioned as possible molecular targets through which drugs could induce cytotoxicity. These cell-based systems allow assessment of drug-channel interaction by monitoring the effect of the drug on currents produced by different channels in cultured cells using a technique known as “patch clamping.” Patch clamping isolates regions of the cell membrane containing channel proteins and measures changes in electrical potential difference. However, use of this method in high throughput requires automation of patch clamping in an array format with reliable giga seal, which even though is becoming increasing available, is not yet widespread. In addition, cardiac toxicity may occur by other mechanisms that could be possibly missed by this type of targeted approach.
An alternative to in vitro ion-channel recording assays as well as the labor-intensive isolation of primary tissue is the differentiation of embryonic stem (ES) cells into cardiomyocytes. The utility of ES cells as a treatment for various chronic diseases has received much attention in recent years. Mammalian ES cells are self renewing cells derived from the inner cell mass of a blastocyst stage embryo which can be differentiated into multiple different cell types. It has been demonstrated that the mouse ES cells as well as human ES cells can be differentiated into cardiomyocytes which retain the ability to beat in culture. Differentiation of ES cells first involves an intermediate in vitro developmental stage in which ES cells form compact cell structures known as embryoid bodies. These embryoid bodies can induce the developmental program of ES cell differentiation into multiple cell types including cardiomyocytes, which are distinguished in culture by their ability to undergo spontaneous beating. These ES derived in vitro differentiated cardiomyocytes recapitulates the normal development of cardiomyocytes as evidenced by the stage-specific expression of cardiomyocyte specific genes. All the known transcription factors, ion channels and structural proteins that are part of normal heart development and function in vivo are also expressed in ES-derived cardiomyocytes.
Even though high throughput to medium throughput systems have been developed for functional characterization of cell lines heterologously expressing the gene for specific ion channels, high throughput techniques for functional characterization of more complex systems such as cardiomyocytes have been limited. Technologies designed to assess cardiomyocyte behaviour and function and the effect of drugs and other manipulations in vitro can be divided into two different approaches. One approach involves long term assessment of cardiomyocyte viability for example in response to certain compounds. Such assays are typically end point assays designed to measure a cellular component such as ATP which correlates with the degree of viability of the cells. The other approach involves studying short term effect of drugs and compounds on beating function of cardiomyocytes. High throughput techniques for short term functional characterization of ion channels and other targets in cardiomyocytes has been rather challenging and limited. The available systems typically only monitor a single cardiomyocyte or a small number of cardiomyocytes at a time with very limited throughput.